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J. Biol. Chem., Vol. 275, Issue 42, 33091-33101, October 20, 2000

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Isolation and Characterization of IIA^Chb, a Soluble Protein of the Enzyme II Complex Required for the Transport/Phosphorylation of N,N'-Diacetylchitobiose in Escherichia coli^*

Nemat O. Keyhani, Olga Boudker^§, and Saul Roseman^¶

From the Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

N,N'-Diacetylchitobiose is transported/phosphorylated in Escherichia coli by the (GlcNAc)₂-specific Enzyme II permease of the phosphoenolpyruvate:glycose phosphotransferase system. IIA^Chb, one protein of the Enzyme II complex, was cloned and purified to homogeneity. IIA^Chb and phospho-IIA^Chb form stable homodimers (3). Phospho-IIA^Chb behaves as a typical 2-N (i.e. N-3) phospho-His protein. However, the rate constants for hydrolysis of phospho-IIA^Chb at pH 8.0 unexpectedly increased 7-fold between 25 and 37 °C and increased ~ 4-fold with decreasing protein concentration at 37 °C (but not 25 °C). The data were explained by thermal denaturation studies using CD spectroscopy. IIA^Chb and phospho-IIA^Chb exhibit virtually identical spectra at 25 °C (~80% -helix), but phospho-IIA^Chb loses about 30% of its helicity at 37 °C, whereas IIA^Chb shows only a slight change. Furthermore, the T_m for thermal denaturation of IIA^Chb was 54 °C, only slightly affected by concentration, whereas the T_m for phospho-IIA^Chb was much lower, ranging from 40 to 46 °C, depending on concentration. In addition, divalent cations (Mg²⁺, Cu²⁺, and Ni²⁺) have a dramatic and differential effect on the structure, depending on the state of phosphorylation of the protein. Thus, phosphorylation destabilizes IIA^Chb at 37 °C, potentially affecting the monomer/dimer transition, which correlates with its chemical instability at this temperature. The physiological consequences of this phenomenon are briefly considered.

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